Project Summary: RNA therapeutics represents a new class of modern medicine for targets considered undruggable. Nanoparticle based platforms remain the most advanced in clinical trials for RNA based drugs. Yet, the lack of mechanistic insights into the cellular trafficking and endosomal escape of nanoparticles has become a major hurdle for efficient intracellular delivery. Nanoparticles enter cells through highly dynamic endocytic pathways that are routed towards lysosomes for degradation. This study aims to 1) Determine the gateways of cellular entry and subsequent itinerary of lipid nanoparticles (LNP) that deliver messenger RNA (mRNA) inside cells through the use of state of the art microscopy techniques in combination with different markers of endocytosis and/or inhibitors of select trafficking pathways 2) Dissect the productive sites for endosomal escape by utilizing an CRISPR/Cas9 and/or shRNA based library targeted against endosomal proteins, that direct nanoparticles towards the early, recycling, late or lysosomal routes of delivery. The disruption of key steps in endocytic trafficking will trigger release of nanoparticles from vesicular confinement and reveal the active sites for endosomal escape 3) Identify bioactive lipids that improve escape from productive endocytic compartments. These lipids were selected based on their properties of influencing cell membrane dynamics, cell signaling and enrichment into the endo/lysosomal system that can trigger endosomal escape. In these studies custom-built 3D stochastic optical reconstruction microscopy (3D-STORM) and 3D multi-resolution microscopy (3D-MM) will be employed to visualize endosomal escape and identify triggers that improve cytosolic delivery. Our preliminary investigation using super-resolution microscopy reveals transport of LNP delivered nucleic acids with very high spatiotemporal resolution. Using genetically altered cells we were able to pinpoint key stages of LNP mediated mRNA delivery. Novel bioactive lipids that can improve intracellular delivery of mRNA have also been identified and are being interrogated for their ability to breach endosomal barriers. Our goal is to unlock the mechanisms of carrier-mediated intracellular delivery, unravel productive sites of endosomal escape and identify bioactive lipids that can enable intracellular mRNA delivery that will, in the future, lead to efficient production of therapeutic proteins for the treatment of various devastating disorders. Our long-term plan is to build a firm basic foundation, which enables further development and optimization of novel nanoparticles to overcome cellular barriers and reach drug targets.